1. Field of the Invention
The present invention relates generally to instrumentation and methods for manipulating membrane potentials of living cells via electrical stimulation.
2. Description of the Related Art
It has long been known that the interior of animal and plant cells is electrically negative with respect to the exterior. The magnitude of this potential difference is generally between 5 and 90 mV, with most of the potential being developed across the cell membrane. The transmembrane potential of a given cell is set by the balance of the activities of ion transporters which create and maintain the electrochemical gradient, and the activities of ion channels, passive diffusion and other factors, that allow ions to flow through the plasma membrane.
Ion channels participate in, and regulate, cellular processes as diverse as the generation and timing of action potentials, energy production, synaptic transmission, secretion of hormones and the contraction of muscles, etc. Many drugs exert their specific effects via modulation of ion channels. Examples include antiepileptic compounds like phenytoin and lamotrigine, which block voltage-dependent sodium channels in the brain, antihypertensive drugs like nifedipine and diltiazem, which block voltage-dependent calcium channels in smooth muscle cells, and stimulators of insulin release like glibenclamide and tolbutamide, which block ATP-regulated potassium channels in the pancreas.
Finding new drugs which have specific modulatory effects on ion channels requires methods for measuring and manipulating the membrane potential of cells with the ion channels present in the membrane. A number of methods exist today that can be used to measure cell transmembrane potentials and to measure the activities of specific ion channels. Probably the best known approach is the patch clamp, originally developed by Neher, Sakmann, and Steinback. (The Extracellular Patch Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranesxe2x80x9d, Pfluegers Arch. 375; 219-278, 1978). Other methods include optical recording of voltage-sensitive dyes (Cohen et al., Annual Reviews of Neuroscience 1:171-82, 1978) and extracellular recording of fast events using metal (Thomas et al., Exp. Cell Res. 74:61-66, 1972) or field effect transistors (FET) (Fromherz et al., Science 252:1290-1293, 1991) electrodes.
The patch clamp technique allows measurement of ion flow through ion channel proteins and the analysis of the effect of drugs on ion channels function. In brief, in the standard patch clamp technique, a thin glass pipette is heated and pulled until it breaks, forming a very thin ( less than 1 xcexcm in diameter) opening at the tip. The pipette is filled with salt solution approximating the intracellular ionic composition of the cell. A metal electrode is inserted into the large end of the pipette, and connected to associated electronics. The tip of the patch pipette is pressed against the surface of the cell membrane. The pipette tip seals tightly to the cell and isolates a few ion channel proteins in a tiny patch of membrane. The activity of these channels can be measured electrically (single channel recording) or, alternatively, the patch can be ruptured allowing measurements of the combined channel activity of the entire cell membrane (whole cell recording).
During both single channel recording and whole-cell recording, the activity of individual channel subtypes can be further resolved by imposing a xe2x80x9cvoltage clampxe2x80x9d across the membrane. Through the use of a feedback loop, the xe2x80x9cvoltage clampxe2x80x9d imposes a user-specified potential difference across the membrane, allowing measurement of the voltage, ion, and time dependencies of various ion channel currents. These methods allow resolution of discrete ion channel subtypes.
A major limitation of the patch clamp technique as a general method in pharmacological screening is its low throughput. Typically, a single, highly trained operator can test fewer than ten compounds per day using the patch clamp technique. Furthermore the technique is not easily amenable to automation, and produces complex results that require extensive analysis by skilled electrophysiologists. By comparison, the use of optical detection systems provides for significantly greater throughput for screening applications (currently, up to 100,000 compounds per day), while at the same time providing for highly sensitive analysis of transmembrane potential. Methods for the optical sensing of membrane potential are typically based on translocation, redistribution, orientation changes, or shifts in spectra of fluorescent, luminescent, or absorption dyes in response to the cellular membrane potential (see generally Gonzxc3xa1lez, et al., Drug Discovery Today 4:431-439, 1999).
A preferred optical method of analysis has been previously described (Gonzxc3xa1lez and Tsien, Chemistry and Biology 4:269-277, 1997; Gonzxc3xa1lez and Tsien, Biophysical Journal 69:1272-1280, 1995; and U.S. Pat. No 5,661,035 issued Aug. 26, 1997, hereby incorporated by reference). This approach typically comprises two reagents that undergo energy transfer to provide a ratiometric fluorescent readout that is dependent upon the membrane potential. The ratiometric readout provides important advantages for drug screening including improved sensitivity, reliability and reduction of many types of experimental artifacts.
Compared to the use of a patch clamp, optical methods of analysis do not inherently provide the ability to regulate, or clamp, the transmembrane potential of a cell. Clamping methods are highly desirable because they provide for significantly enhanced, and more flexible methods of ion channel measurement. A need thus exists for reliable and specific methods of regulating the membrane potentials of living cells that are compatible with optical methods of analysis and are readily amendable to high throughput analysis.
Methods and systems of compound screening are provided. In one embodiment, such a method comprises expressing the target ion channel in a population of host cells and placing a plurality of the host cells into each of a plurality of sample wells. A candidate drug compound is added to at least one of the plurality of sample wells; and the transmembrane potential of the cells is modulated with a repetitive application of electric fields so as to set the transmembrane potential to a level corresponding to a pre-selected voltage dependent state of the target ion channel. Apparatus for high throughput screening is also provided. In one specific embodiment, a plurality of wells having a high transmittance portion through which cells present in the wells are optically observable in an area of observation are each provided with two electrodes. A power supply is connected to the electrodes; wherein the power supply and the electrodes are configured to apply a series of electric fields to cells within the area of observation, the electric fields having a spatial variation of less than about 25% of a mean field intensity within the area of observation, the electric fields being effective to controllably alter the transmembrane potential of a portion of the cells. In addition, an optical detector is configured to detect light emanating from the wells through the high transmittance portion, and a data processing unit is provided to interpret the light emanating from the wells through the high transmittance portion as ion channel activity resulting from the transmembrane potential alterations.